Identification of Biomarkers of Cardiotoxicity using Metabolomics of Human Pluripotent Stem Cell- Derived Cardiomyocytes Project Summary/Abstract Cardiac safety is one of the leading causes of compound attrition in the pharmaceutical industry and withdrawal of FDA-approved drugs from the market. The purpose of this proposal is to improve public health, as well as alleviate the financial burden of compound attrition due to cardiotoxicity, through development of an in vitro assay to predict a compound's cardiotoxicity potential. To accomplish this, Stemina Biomarker Discovery (Stemina) proposes to use metabolomics of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to known human cardiotoxic and non-cardiotoxic compounds. These technologies will be used to discover human, endogenous metabolite biomarkers which predict general cardiotoxicity as well as the specific type of cardiotoxicity (e.g., functional, structural). The use of metabolomics to measure small molecules secreted by hiPSC-CMs in response to compound exposure is a novel approach for evaluating cardiotoxicity and may pave the way for a new generation of more accurate, predictive toxicology screens using human cells. Stemina already used such a paradigm to complete the Phase I SBIR Application (1R43GM100640-01), as well as developed predictive methods to assess developmental toxicity potential in undifferentiated pluripotent stem cells (devTOX(tm)). Stemina's long-term goal is to develop a human cell- based, high-throughput cardiotoxicity screen as a valuable tool to pharmaceutical, biotech, and agrichemical companies during early development of therapeutics and chemicals. In order to achieve this goal, Stemina first proposes to develop an optimized and reproducible experimental platform to evaluate spent media collected from hiPSC-CMs (aim 1). We will evaluate various sample preparation methods, LC-MS columns and conditions, and perform robustness testing in order to establish the most reproducible measurement of the complete set of secreted metabolites, or secretome, in hiPSC-CMs using our system. In aim 2, we will use the above platform to evaluate spent media from hiPSC- CMs response to a training set of 60 compounds consisting of functional-, structural-, general-, and non- cardiotoxicants to establish a predictive metabolomic model. Small molecules whose abundances vary dependent upon whether cells were treated with an inducer or non-inducer of cardiotoxicity will serve as candidate biomarkers of cardiotoxicity. The data acquired here will be used to establish a predictive metabolic signature indicative of general cardiotoxicity and specific type of cardiotoxicity (e.g., functional, structural). Stemina will then test the performace of the predictive model(s) of biomarker signature(s) on a test set of 20 compounds. In aim 3, Stemina will confirm the structural identity of the predictive metabolites and evaluate their biological significance as confirmed biomarkers. Lastly in aim 4, a targeted biomarker assay will be developed using targeted LC-MS methods that measure the confirmed biomarkers. Further, the ability of our biomarkers to adequately predict cardiotoxicity will be tested through the use o a blind study comprised of compounds acquired from partnering companies. Completion of these aims will enable the development of a commercial assay able to detect the validated biomarkers of cardiotoxicity, similar to an existing test Stemina currently markets for developmental toxicity (devTOX(tm) quickPredict). Stemina will subsequently utilize this assay to market a service capable of predicting whether a compound will induce cardiotoxicity and serve pharmaceutical companies in preclinical screening trials. Such a service provides the first human cell-based screening assay for cardiotoxicity founded on cardiomyocyte metabolism.